The goal of this proposal is to investigate the therapeutic potential of transcutaneous vagus nerve stimulation (tVNS) in acute cerebral ischemia. Electrical stimulation of the afferent vagal fibers in the neck has been shown to activate the brainstem vagal centers, induce anti-excitotoxic and anti-inflammatory mechanisms, and increase cerebral blood flow (CBF). Stimulation of the cervical vagus nerve using surgically implanted electrodes (cVNS) is an FDA-approved treatment in some epilepsy and depression patients. Recent studies demonstrated that cVNS provides substantial protection against tissue injury and motor function loss in animal models of ischemic stroke as well. cVNS, when initiated up to 2.5 hours after ischemia, reduces infarct volume by approximately 50% in a variety of rat models of focal cerebral ischemia and in animals with comorbid conditions. The mechanism of protection is not clear yet, but initial findings suggest that it is CBF independent. Even though the findings of ischemia studies are quite encouraging, cVNS is not applicable to acute stroke cases in humans due to invasive nature of the stimulation. In this application, we propose two different tVNS approaches to circumvent the problem of surgical intervention: (1) to stimulate the cervical vagus nerve through the intact skin using a recently available technology and (2) to stimulate the auricular branch of the vagus nerve that supplies a dermatome in external ear using needle electrodes. Both cervical and auricular tVNS have been safely and successfully used in patients with hiccups and pain, respectively. Our specific aims are: (1) determine the efficacy of tVNS techniques in focal ischemia, (2) describe the optimal time window of tVNS treatment, (3) study whether concomitant application of tVNS and tPA is safe and effective in cerebral ischemia. We will determine the effects of cervical tVNS and ear tVNS on infarct size (as assessed by vital stain) and motor function (as assessed by behavioral tests and grip strength measurements). To determine their safety, we will monitor physiological parameters (e.g., arterial blood pressure, heart rate, heart rate variability, and respiration rate) during an immediately after stimulation and will follow-up animals up to 1 week after the treatment. In an additional group of animals, to verify the activation of brainstem vagal centers, we will perform c Fos immunohistochemistry after tVNS. After determining the safety and efficacy of neck tVNS and ear tVNS in specific aim 1, we will continue our studies with the safest and most efficacious tVNS. To determine the time window of tVNS after ischemia, we initiate tVNS 3 hours or more after ischemia and determine its effect on infarct size. We also propose to determine the interaction between tPA and tVNS. We will examine the effect of tPA - tVNS co-administration on risk of hemorrhage. We also will investigate the effect of tVNS on tPA's time window and efficacy using multi-modal MR imaging with molecular thrombus imaging. The proposed studies will provide the proof of principle that tVNS improves stroke outcome. If successful, the findings of these exploratory studies will be used in near future translational studies designed to optimize tVNS treatment for acute ischemic stroke in humans.